Wang Zhonghua, Ando Yuki, Nugraheni Ari Dwi, Ren Chunguang, Nagao Satoshi, Hirota Shun
Graduate School of Materials Science, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan.
Mol Biosyst. 2014 Dec;10(12):3130-7. doi: 10.1039/c4mb00285g.
Met80 of cytochrome c (cyt c) has been shown to dissociate from its heme iron when cyt c interacts with cardiolipin (CL), which triggers the release of cyt c into the cytosol initiating apoptosis. We found that the mass of human cyt c increases by 16 Da in the Met80-Lys86 region by reaction with molecular oxygen in the presence of CL-containing liposomes and dithiothreitol (DTT). To investigate the effect of Met80 dissociation on the reaction of cyt c with molecular oxygen without affecting its secondary structures, a human cyt c mutant (Δ8384 cyt c) was constructed by removing two amino acids (Val83 and Gly84) from the loop containing Met80. According to MALDI-TOF-MS and tandem mass measurements, Met80 of Δ8384 cyt c was modified site-specifically to methionine sulfoxide when purified in the presence of molecular oxygen, whereas Met80 was not modified in the absence of molecular oxygen. A red-shift of the Soret band from 406 to 412 nm and absorption increase at ∼536 and ∼568 nm were observed for Δ8384 cyt c when it reacted with DTT and molecular oxygen, followed by a further red-shift of the Soret band to 416 nm and absorption increase at ∼620 and ∼650 nm. These results indicate that Met80 of cyt c is oxidized site-specifically by formation of the oxy and subsequent compound I-like species when Met80 dissociates from the heme iron, where the Met80 modification may affect its peroxidase activity related to apoptosis.
细胞色素c（cyt c）的甲硫氨酸80（Met80）已被证明在与心磷脂（CL）相互作用时会与其血红素铁解离，这会触发细胞色素c释放到细胞质中从而引发细胞凋亡。我们发现，在含有CL的脂质体和二硫苏糖醇（DTT）存在的情况下，人细胞色素c通过与分子氧反应，其Met80-Lys86区域的质量增加了16 Da。为了研究Met80解离对细胞色素c与分子氧反应的影响而不影响其二级结构，通过从包含Met80的环中去除两个氨基酸（缬氨酸83和甘氨酸84）构建了人细胞色素c突变体（Δ8384 cyt c）。根据基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）和串联质谱测量，当在分子氧存在下纯化时，Δ8384 cyt c的Met80被位点特异性修饰为甲硫氨酸亚砜，而在没有分子氧的情况下Met80未被修饰。当Δ8384 cyt c与DTT和分子氧反应时，观察到其Soret带从406 nm红移至412 nm，并且在约536和约568 nm处吸收增加，随后Soret带进一步红移至416 nm，并且在约620和约650 nm处吸收增加。这些结果表明，当Met80与血红素铁解离时，细胞色素c的Met80通过形成氧和随后的类似化合物I的物种而被位点特异性氧化，其中Met80修饰可能影响其与细胞凋亡相关的过氧化物酶活性。
